Bioresource Technology 245 (2017) 1674–1683

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Bioresource Technology

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Engineering aspects of microbial exopolysaccharide production ⇑ Filomena Freitas, Cristiana A.V. Torres, Maria A.M. Reis

UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal

highlights graphical abstract

 Microbial EPS producers: bacteria, fungi and microalgae.  EPS production by extremophiles.  Mixed microbial consortia: alginate- like EPS.  Cultivation mode and process operation conditions.  Different bioreactor design for microbial EPS production.

article info abstract

Article history: Although the ability to secrete exopolysaccharides (EPS) is widespread among microorganisms, only a Received 30 March 2017 few bacterial (e.g. xanthan, levan, ) and fungal (e.g. pullulan) EPS have reached full commercial- Received in revised form 13 May 2017 ization. During the last years, other microbial EPS producers have been the subject of extensive research, Accepted 15 May 2017 including endophytes, extremophiles, microalgae and Cyanobacteria, as well as mixed microbial consor- Available online 18 May 2017 tia. Those studies have demonstrated the great potential of such microbial systems to generate biopoly- mers with novel chemical structures and distinctive functional properties. In this work, an overview of Keywords: the bioprocesses developed for EPS production by the wide diversity of reported microbial producers Exopolysaccharide (EPS) is presented, including their development and scale-up. Bottlenecks that currently hinder microbial Bacteria Fungi EPS development are identified, along with future prospects for further advancement. Extremophiles Ó 2017 Elsevier Ltd. All rights reserved. Mixed microbial consortia

1. Introduction few of them, namely, , levan, dextran and pullulan, that are well known industrial with considerable Microbial exopolysaccharides (EPS) are the research subject of markets (Freitas et al., 2011a; Moscovici, 2015). many scientific areas, with special focus on optimization of their Compared to other natural sources, microbial EPS usually have production processes, elucidation of their function for the produc- considerable shorter production times and their extraction is much ing organisms, identification of the biosynthetic pathways involved simpler. Microbial bioprocesses are performed in fermenters, not in their synthesis and the development of applications based on competing with food production lands, and it is possible to use their distinct properties. Within these research areas, a huge diver- wastes as feedstocks. Furthermore, those polysaccharides present sity of microbial EPS structures have been reported but the poten- a greater variety of structures and physicochemical properties, ren- tial for their industrial development has been examined for only a dering them applicability in areas ranging from their use as bioma- terials or as rheology modifiers of aqueous systems, to therapeutic agents (Donot et al., 2012; Moscovici, 2015; Rehm, 2010). ⇑ Corresponding author at: UCIBIO-REQUIMTE, Departamento de Química, There is a growing number of recent reports on newly discov- Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, 2825-516 Caparica, Portugal. ered EPS secreted by producers belonging to different taxonomic E-mail address: [email protected] (M.A.M. Reis). groups that have novel molecular structures. Therefore, the

http://dx.doi.org/10.1016/j.biortech.2017.05.092 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved. F. Freitas et al. / Bioresource Technology 245 (2017) 1674–1683 1675 processes for production of these natural biopolymers hold great Alginate is a linear anionic polymer composed of b-D- potential for development, although their industrialization faces mannuronic acid (M) and a-L-guluronic acid (G). The monomers many challenges and proceeds at a slow pace. may be arranged in MM, GG or MG blocks (Mejía et al., 2009) and are acetylated (Galindo et al., 2007). Alginates are secreted by bacteria of the Genera Pseudomonas and Azotobacter. 2. Microbial EPS producers and products Levan is a branched homofructan (Öner et al., 2016; Srikanth et al., 2015b). It is synthesized from by the action of levan- Microbial EPS producers include both eukaryotes (phytoplank- sucrase, an enzyme secreted by a variety of bacteria, including spe- ton, fungi, algae) and prokaryotes (eubacteria and archaebacteria) cies of the genera Halomonas, Zymomonas, Bacillus and (Delattre et al., 2016; Donot et al., 2012; Moscovici, 2015; Pseudomonas (Öner et al., 2016)(Table 1). Levan is soluble in water Osinska-Jaroszuk et al., 2015; Rehm, 2010)(Table 1). Although still and oil, compatible with salts and surfactants, and has emulsifying not completely understood, several important physiological func- capacity, biological activity, film forming and adhesive capacity, tions have been proposed for microbial EPS, including protection that turns it suitable for use in several applications (Silbir et al., against environmental pressures (e.g. osmotic stress, temperature 2014; Zhang et al., 2014). or pH, damage by UV light, heavy metals, oxidants, desiccation), Sphingomonas paucimobilis ATCC 31461 is used for the indus- cell adherence to surfaces and carbon or water storage reserves trial production of gellan gum, a linear anionic EPS composed of (Delattre et al., 2016; Donot et al., 2012; Moscovici, 2015; a backbone with , and glucuronic Papinutti, 2010; Rehm, 2010). acid residues, and side chains of glyceryl and acetyl substituents (Table 1). Gellan gum was approved by the FDA and is used in sev- 2.1. Bacteria eral applications, such as food, cosmetics, dental care and biomedi- cine. It is commercialized under the trade names Kelcogel, Gelrite The ability to secrete polysaccharides is broadly distributed and Gel-Gro (Prajapati et al., 2013a). among bacteria, including species of the Genera Streptococcus, Xan- In addition, there are numerous other bacteria (e.g. Klebsiella thomonas and Acetobacter (Table 1). The first bacterial EPS to be pneumoniae, Pseudomonas oleovorans, Enterobacter A47, Escherichia commercialized was dextran, a water soluble produced by coli, etc.) reported to secrete EPS with distinctive structures and of the genera Leuconostoc, Streptococcus, Wei- novel properties (Table 1)(Freitas et al., 2009, 2011b; Patel and sella, Pediococcus and Lactobacillus, which are Generally Regarded Prajapati, 2013). Many bacteria isolated from extreme habitats as Safe (GRAS) and commonly used in different food products man- (e.g. Antarctic ecosystems, saline lakes, geothermal springs), like ufacturing (Patel and Prajapati, 2013). Curdlan is another glucan Pseudoalteromonas sp., Bacillus sp., Halomonas sp., and Vibrio sp., produced by Alcaligenes and Agrobacterium species (Table 1). It is were also identified as EPS producers. Most of the EPS contained a water insoluble gel-forming polymer. Both dextran and curdlan glucose and/or and charged uronic acid residues. Pseu- were approved by the FDA (Food and Drug Administration), being doalteromonas sp. SM9913 produces an EPS with flocculating and used in food, cosmetic and medical applications (Patel and metal binding capacity (Li et al., 2008). Several Halomonas strains Prajapati, 2013; Yang et al., 2016). isolated from hyper saline zones were reported to produce EPS Some bacteria, including species of the Genera Acetobacter, Glu- containing high contents of sulfate and uronic acids. The deep canoacetobacter and Rhizobium, secrete , a water insoluble sea bacteria Vibrio diabolicus produces a HA-like EPS that presents biopolymer with a high mechanical strength and low density, and great potential in vivo regeneration of tissues and the ability to high water adsorption capacity (Chawla et al., 2009; Gomes et al., accelerate in vitro collagen fibrillation (Senni et al., 2011). This 2013)(Table 1). A. xylinum is the most extensively studied strain EPS is commercialized as HyaluriftÒ by Seadev-FermenSys and and considered for industrial production of bacterial cellulose Infremer. Examples of reported thermophilic EPS producers (BC) (Chawla et al., 2009), but G. sacchari has also shown a good include B. licheniformis strain T4 that synthesizes a fructo-fucan production capacity (Gomes et al., 2013). with anti-cytotoxic and antiviral activity (Gugliandolo et al., Xanthomonas sp. produce xanthan (Table 1), a heteropolysac- 2013; Spanò et al., 2013) and Aeribacillus pallidus 418, which pro- charide composed of a glucose backbone with side duces a complex with emulsifying capacity chains consisting of two mannose units alternating with glu- (Radchenkova et al., 2014). curonic acid. Around half of the terminal mannose residues are pyruvated and the non-terminal residues are linked to acetyl 2.2. Fungi and yeasts groups. Commercially, it is produced by X. campestris (Silva et al., 2009). In 1969, xanthan was approved by the FDA for food use. EPS production is widely distributed among fungi, including Currently, it is the only significant microbial biopolymer in the glo- members of the Genera Aureobasidium, Candida, Cryptococcus, bal hydrocolloids market (Freitas et al., 2011a), marketed with dif- among many other (Table 1). Most of them are homopolysac- ferent purity grades for applications ranging from food to charides but heteropolysaccharides are also very common. Such pharmaceuticals and oil recovery. It is commercialized by Sanofi- biopolymers contain D-mannose, either alone or in combination Elf, Merck, Pfizer, Jungbunzlauer, and Rhône Poulenc (Silva et al., with other (e.g. , ), together with uronic 2009). acids and non- substituents (Table 1)(Osinska-Jaroszuk Streptococcus zooepidemicus produces hyaluronic acid (HA) et al., 2015). Despite the large chemical and structural diversity (Table 1)(Amado et al., 2016), a linear polymer composed of found among fungal EPS, and their reported biological activity, repeating units of glucuronic acid and they are still understudied. The main exception is pullulan, an N-acetylglucosamine (Vásquez et al., 2009; Jia et al., 2013), that EPS secreted by the black yeast-like Aureobasidium pullulans. is used in cosmetics, pharmaceuticals and medicine (Liu et al., Pullulan is a linear a-glucan (Table 1) with valuable properties 2011; Benedini and Santana, 2013). The production by fermenta- that include high mechanical strength and adhesiveness (Cheng tion of Streptococcus zooepidemicus was explored as an alternative et al., 2011; Prajapati et al., 2013b). It is commercialized by to extraction from animal tissues (Jia et al., 2013). The concern Hayashibara Co., Ltd. (Japan), as a thickening agent and edible about the pathogenicity of Streptococcus has driven the efforts coating. towards transforming GRAS non-producers (e.g. L. lactis, B. subtilis) Scleroglucan is a water soluble b-glucan secreted by the plant into HA producers (Jia et al., 2013). pathogen Sclerotium sp. (Schmid et al., 2011; Taskin et al., 2010) 1676 F. Freitas et al. / Bioresource Technology 245 (2017) 1674–1683

Table 1 Microbial EPS producers: bacteria, fungi and microalgae (Glc, glucose; Gal, galactose; Man, mannose; Fru, ; Rha, rhamnose; Fuc, , Ara, ; Xyl, xylose; Rib, ; GlcA, glucuronic acid; GalA, galacturonic acid; ManA, mannuronic acid; GulA, guluronic acid; GlcN, glucosamine; GalN, galactosamine; Ac, acetate; Pyr, pyruvate; Succ, succinate; Glyc, glycerate; Phosp, phosphate; Sulf, sulfate).

Microorganism Polymer Sugar monomers Non-sugar Mw (Da) References residues Bacteria Agrobacterium sp. Curdlan Glc – 5.0 Â 104–2.0 Â 106 Yang et al. (2016) Alcaligenes sp. Azotobacter vinelandii Alginate GulA, ManA Ac (0.3À1.3) Â 106 Cimini et al. (2012), Galindo et al. Pseudomonas aeruginosa (2007) Bacillus subtilis Levan Fru – (1.0À3.0) Â 106 Öner et al. (2016) Halomonas smyrensi Zymomonas mobilis Enterobacter A47 FucoPol Fuc, Gal, Glc, GlcA Ac, Pyr, Succ (2.0À10) Â 106 Freitas et al. (2011b) Klebsiella pneumoniae Fucogel GalA, Fuc, Gal – 3.1 Â 106 Freitas et al. (2011b) Acetobacter sp.; Bacterial Glc – 106 Chawla et al. (2009) Glucanoacetobacter sp. cellulose Rhizobium sp.; Sarcina sp. Lactobacillus sp. Dextran Glc – 1.5 Â 104–2.0 Â 107 Patel and Prajapati (2013) Leuconostoc sp. Streptococcus sp. Pseudomonas oleovorans GalactoPol Gal, Glc, Man, Rha Ac, Pyr, Succ (1.0À5.0) Â 106 Freitas et al. (2009) Sphingomonas paucimobilis Gellan Glc, Rha, GlcA Glyc, Ac Fialho et al. (2008), Prajapati et al. (2013a,b) Streptococcus zooepidemicus Hyaluronic GlcNAc, GlcA – (1.0À2.0) Â 106 Zhang et al. (2006) acid Xanthomonas sp. Xanthan Glc, Man, GlcA Pyr, Ac 2.0 Â 106–5.0 Â 107 Palaniraj and Jayaraman (2011) Fungi Antrodia cinnamomea – Fuc, GlcN, Gal, Glc, Man Sulf 1.0 Â 103–1.0 Â 105 Cheng et al. (2012) Aspergillus sp. Y16 – Man, Gal – 1.5 Â 104 Chen et al. (2011) Aureobasidium pullulans Pullulan Glc – 1.0 Â 106–5.6 Â 106 Prajapati et al. (2013b) and Wu et al. (2016) Diaporthe sp. – Glc, Gal, Man – 4.0 Â 104 Orlandelli et al. (2017) – Glc – 5.2 Â 106 Orlandelli et al. (2017) Fusarium solani – Rha, Gal – 1.9 Â 105 Mahapatra and Banerjee (2012) Ganoderma lucidum – Gal, Man, Glc, Ara, Rha – (2.2–7.8) Â 104 Pan et al. (2012) Sclerotium sp. Scleroglucan Glc – 1.3 Â 105–6.0 Â 106 Castillo et al. (2015), Schmid et al. (2011) Schizophyllum commune Glc – 6.0 Â 106–12.0 Â 106 Zhang et al. (2013) Microalgae Anabaena augstmalis – Glc, Gal, Man, Xyl, Fuc, Rha, GalN, Sulf n.a. Di Pippo et al. (2013) GlcN, GalA, GlcA Dunaliella tertiolecta – Glc – n.a. Goo et al. (2013) Gyrodinium impudicum – Gal Sulf 1.9 Â 106 Yim et al. (2007) Phormidium autumnale – Rha, Rib, Man, Glc, Fuc, Gal, Ara, GalA, Sulf n.a. Di Pippo et al. (2013) GlcA Porphyridium sp. – Xyl, Gal, Glc, GlcA Sulf 2.4 Â 105–1.8 Â 106 Soanen et al. (2016) Rhodella sp. – Xyl, Gal, Glc, Rha, Ara, GlcA Sulf n.a. Villay et al. (2013) Synechocystis aquatilis – Fuc, Glc, Rha, Xyl, Man, GlcN, GalA, Sulf n.a. Di Pippo et al. (2013) GlcA

(Table 1). Scleroglucan was first commercialized in the 70’s, being 2.3. Microalgae currently available under different trademarks (e.g. Clearogel, Polytetran, Polytran FS, Actigum), for use in applications such as Microalgae production is an emergent market with an expected enhanced oil recovery, food, cosmetic and pharmaceutical world yearly growth of 10%. They are used for the production of (Castillo et al., 2015; Taskin et al., 2010). livestock feed and fish meal, organic fertilizers, and high value Many higher fungi belonging to the Genera Ganoderma, Poria, extracts, such as natural pigments, nutraceuticals and antioxi- Tremella (Table 1) secrete EPS with bioactive properties that dants. The main species produced are Arthrospira platensis, Aphani- include antioxidant, antitumor, immunostimulating and antimi- zomenon, Chlorella vulgaris, Dunaliella salina and Porphyridium crobial properties (Osinska-Jaroszuk et al., 2015). According to cruentum. Microalgae EPS are characterized by complex chemical recent reports, endophytic fungi synthesize EPS that are involved structures, ranging from homopolymers of glucose or galactose, in plant-endophyte interactions and such biopolymers are charac- to heteropolysaccharides composed of several sugar monomers terized by novel structures exhibiting biological activity (Liu et al., (Table 1). Compared to other microbial producers, microalgae syn- 2017). Examples include the galactomannan produced by Aspergil- thesize EPS with a higher diversity of sugar monomers in the same lus sp. Y16, which was reported to have antioxidant activity (Chen macromolecule. Of notice is also the presence of rare sugars, such et al., 2011), the b- produced by Diaporthe sp. strains as fucose, rhamnose and ribose. The presence of uronic acids and JF766998 and JF767007 that have antitumor activity (Orlandelli sulfate is very common among algal EPS, which further contributes et al., 2017), and the rhamnogalactan produced by Fusarium solani to their distinctive properties. SD5 that exhibited in vitro anti-inflammatory and anti-allergic Although several microalgae have been reported to secrete activity (Mahapatra and Banerjee, 2012)(Table 1). large amounts of EPS, they are often seen as by-products of other F. Freitas et al. / Bioresource Technology 245 (2017) 1674–1683 1677 productions, such as pigments or lipids (Delattre et al., 2016). can reach high productivity values (up to 18 and 13 g/L.day, Therefore, microalgae EPS are still largely unexploited and only respectively). On the other hand, low productivity values (0.06– recently their relevance as producers of valuable polysaccharides 1.32 g/L.day) were reported for EPS production by higher fungi, as started to be considered. Nevertheless, some microalgae EPS such as G. lucidum (Table 3), and even lower for microalgae have already started to be exploited, such as, for example, the (0.003–0.108 g/L.day) (Soanen et al., 2016; Villay et al., 2013). one synthesized by Porphyridium sp. that is marketed by Frutarom Those bioprocesses are mostly still under development and the under the trade name AlguardTM for use in cosmetics. interest in their optimization is supported by the valuable func- tional properties of the EPS that are not found in other polymers. 2.4. Mixed microbial consortia Carbon availability concomitant with limiting nitrogen is usu- ally reported as favoring EPS production by microorganisms Several processes for wastewater treatment involve high cell (Freitas et al., 2011a). Under growth limiting conditions, the carbon density bioreactors where microorganisms are aggregated in the source is derived towards polysaccharide synthesis. However, it is form of granules or biofilms. Among these, granular sludge pro- important to define appropriate substrate ranges since under cer- cesses, where granules are composed of a mixture of microbial spe- tain conditions, EPS synthesis may be hindered. Although the pres- cies embedded in a matrix of extracellular polymeric substances ence of extra nitrogen favors cell growth, it often results in poor (polysaccharides, proteins, lipids), metal ions and various minerals EPS production. For example, cellular growth of Enterobacter A47 (Zhang et al., 2016) are widely used. Some of the microbial species was improved by the increase of initial nitrogen concentration present in granules secrete various kinds of polysaccharides (up to but a reduction of FucoPol production was observed (Torres 10% of the granules’ mass) that act as gelling agents, contributing et al., 2014). Hence, high amounts of biomass do not necessarily to the granules’ structure and properties (Lin et al., 2010). Species lead to optimal EPS production. On the other hand, decreasing of the genus Pseudomonas, Clostridium, Thauera and Arthrobacter the cell growth rate may increase the availability of isoprenoid (Yang et al., 2014) were identified in aerobic granular sludge. Given lipid carrier for non-growth functions, thus stimulating polysac- the high amount of granular sludge produced in different processes charide production (Fialho et al., 2008). In contrast, nitrogen avail- worldwide, there has been recently an increased interest to assess ability stimulates levan production by Bacillus subtilis and the potential for EPS recovery from these systems. Paenibacillus sp. 2H2, because the enzymes required for polymer Alginate-like exopolysaccharides (ALE) were extracted from production are synthesized concomitantly with cell growth aerobic (Lin et al., 2010; Yang et al., 2014) and anaerobic (Li (Esawy et al., 2013; Rütering et al., 2016). Also, curdlan produced et al., 2016a) granular sludge. Lin et al. (2010) reported that ALE by Rhizobium radiobacter was improved by adding 10 g/L (NH4)2- having a high GG content resulted in a more hydrophobic aerobic HPO4 at the end of exponential growth-phase (Wang et al., 2016a). granular sludge. Granulan, a highly complex heteropolysaccharide, Glucose and sucrose are the most commonly used carbon sources with a composition distinct from that of ALE, was recovered from for microbial cultivation and production of EPS for most microorgan- aerobic granular sludge performing N and P removal from isms (Tables 2 and 3). Considering that media costs account for up to nutrient-rich industrial wastewater. It was composed of galactose, 30% of the total costs of fermentative processes (Freitas et al., 2011a), mannose, glucosamine, N-acetyl-galactosamine and 2-acetoa there is an intensive search for cheaper substrates. Several wastes mido-2-deoxy-galactopyranuronic acid, with galactose and glu- and by-products arising from agriculture and food processing indus- curonic acids branches attached to 2-acetoamido-2-deoxy-galacto tries (e.g. cheese whey, waste tomato paste, waste fruit pulp, sugar- pyranuronic acid, and rhamnose branches attached to galactose cane) (Antunes et al., 2015, 2017; Mehta et al., 2014; Survase et al., (Seviour et al., 2012). According to the authors, the synthesis of 2007a), as well as some materials derived from other industries, granulan was induced under conditions that favored the selective such as glycerol by-product (Freitas et al., 2009, 2011b), have been enrichment of Candidatus ‘‘Competibacter phosphatis”. proposed for the production of different microbial EPS. The cost of handling/disposal of waste granular sludge repre- Chen et al. (2013b) reported the use of wheat-straw hydrolysate sents up to 50% of the wastewater treatment costs (Lin et al., by G. xylinus to produce BC, achieving higher production (8.3 g/L) 2015). Hence, the valorization of the waste sludge would con- than with glucose-based medium (4.5 g/L) (Table 2). Olive mill tribute to increase the sustainability and economics of wastewater residues were also tested to produce BC by G. sacchari, although treatment. An example of that is the use of the polysaccharide- lower production yields were achieved (0.81 g/L) (Gomes et al., based biomaterial recovered from granular sludge as a coating 2013). Li et al. (2016b) tested kitchen waste hydrolysate as sub- material for sizing paper or paper-like materials (Lin et al., 2015). strate for xanthan production by X. campestris LRELP-1, achieving a production of 11.73 g/L. Vásquez et al. (2009) used a complex 3. Microbial EPS production processes medium containing peptones from fishing by-products as nitrogen source to produce HA. Although HA productivity was reduced, There is no general cultivation conditions suitable for all micro- there was a cost reduction of 34%. On the other hand, Amado bial producers that will guaranty high EPS productivities and et al. (2016) replaced tryptone by cheese whey supplemented with yields. Bacteria are the most extensively studied producers yeast extract and salts for HA production by S. zooepidemicus and (Table 2). Depending on the species and the cultivation conditions, achieved similar productivities (4.02 g/L) to the ones with syn- EPS production by bacteria may range between 0.29 and 100 g/L, in thetic medium (4.85 g/L) (Table 3). processes taking 0.5–7 days. This corresponds to volumetric pro- Several waste feedstocks were shown to support pullulan pro- ductivity values of 0.19–100 g/L.day (Table 2). The highest values duction at levels (18–84 g/L) comparable to those reported for were reported for levan (59.5–100.0 g/L), curdlan (65.3 g/L) and sucrose (3–39 g/L) and glucose (22–89 g/L) (Table 3). The use waste xanthan (25.0 g/L). Lower productivity values were reached for loquat kernels (Taskin et al., 2010), as well as sugarcane juice, sug- some species (e.g. Glucanoacetobacter sp., L. mesenteroides and S. arcane molasses and coconut water (Survase et al., 2007a), were zooepidemicus) but the polymers have increased value due their also shown to be suitable substrates for scleroglucan production, properties, such as, for example, HA and BC (Table 2). Fungi usually reaching concentrations (12–24 g/L) similar to sucrose (16–26 g/ have longer cultivation times (2–32 days) than bacteria (0.5– L) (Table 3). Such waste substrates often need specific processing 7 days), which in some cases translates into lower volumetric pro- conditions before they can be used in the bioprocesses, which adds ductivities (Tables 2 and 3). Nevertheless, fungi like A. pullulans and to the overall polymer production costs. On the other hand, the use S. rolfsii, the producers of pullulan and scleroglucan, respectively, of such substrates may have a negative impact on the yield and Table 2 1678 EPS production by some bacteria: carbon and nitrogen sources, cultivation conditions and EPS production polymer volumetric productivity (rP) and yield on substrate (Yp/s ) (YE, yeast extract; Pep, peptone; Tryp, tryptone).

Microorganism Process Carbon Nitrogen Time pH T EPS r P Y p/s References source source (d) (°C) (g/L) (g/L.day) (g/g) Agrobacterium sp. Shake flask Sucrose Urea 4.60 7.5 30 5.02 1.10 n.a. Shih et al. (2009) Alcaligenes faecalis Shake flask Glucose Urea 4.00 n.a. 30 24.32 7.04 n.a. Jiang (2013) NH4Cl 5.00 15.17 3.03 Azotobacter vinelandii Shake flask Glucose YE 3.00 7.2 30 3.50 1.17 n.a Moral and Yildiz (2016) Sucrose 3.50 1.17 Molasses 4.67 1.56 Two stage bioreactor Sucrose YE 2.50 7.2 29 9.50 4.32 0.74 Mejía et al. (2009) Bacillus methylotrophycus Shake flask Sucrose n.a. 1.00 6.0 37 100 100.00 0.33 Zhang et al. (2014) Bacillus subtilis Repeated fed-batch bioreactor Sucrose Pep + Beef extract 3.00 5.6–5.8 37 70.60 23.53 n.a. Shih et al. (2010) Batch bioreactor Sucrose YE + Tryp 1.00 7.0- 30 59.5 59.5 Esawy et al. (2013) Enterobacter A47 Fed-batch bioreactor Glycerol by-product (NH4)2HPO4 4.00 7.0 30 7.50–7.97 2.04–2.51 n.a. Freitas et al. (2014) Glucose 13.23 3.38 Xylose 5.39 1.39 Glucanoacetobacter hansenii Shake flask Sucrose KNO3 6.00 5.0 25 5.00 0.83 n.a. Mohite et al. (2013) .Fetse l irsuc ehooy25(07 1674–1683 (2017) 245 Technology Bioresource / al. et Freitas F. Glucanoacetobacter sacchari Shake flask (static conditions) Dry olive mill residue (NH4)2SO4 4.60 4.5 30 0.81 0.19 0.10 Gomes et al. (2013) Glucanoacetobacter xylinus (Acetobacter xylinum) Shake flask Sucrose Pep + (NH4)2SO4 2.50 6.8 28 12.74 5.09 n.a. Srikanth et al. (2015b) Shake flask (static conditions) Glucose YE + Pep 7.00 5.0 30 3.70–4.50 0.53–0.64 n.a. Chen et al. (2013b) Wheat straw hydrolisate 8.30 1.19 Leuconostoc mesenteroides Shake flask Sucrose YE + Pep 0.75 7.5 25 4.89 6.11 0.49 Aman et al. (2012) Pseudomonas sp. Shake flask Sucrose YE 4.00 7.6 30 5.92 1.48 n.a. Yang et al. (2016) Rhizobium radiobacter Fed-batch bioreactor Glucose (NH4)2HPO4 5.00 7.0 30 65.27 13.06 0.39 Wang et al. (2016a) Sphingomonas paucimobilis Batch bioreactor Glucose YE + NH4NO3 1.67 7.0 30 12.30 7.24 n.a. Giavasis et al. (2006) Molasses Tryp n.a. 6.5 30 13.81 n.a. 0.15 Banik et al. (2007) Streptococcus zooepidemicus Batch bioreactor Sucrose YE 0.67 7.2 37 5.00 7.46 0.08 Liu et al. (2008b) Fed-batch bioreactor 4.00 5.97 n.a. Two stage bioreactor 6.60 9.85 0.14 Shake flask Glucose Soy pep 1.00 7.0 37 0.29 0.29 0.04 Benedini and Santana (2013) Fed-batch bioreactor Glucose YE + Tryp 0.33 6.7 30 4.85 14.55 0.07 Vásquez et al. (2009) YE + marine Pep 0.50 1.78–2.14 3.56–4.28 0.66–0.99 Batch bioreactor Cheese whey YE + Tryp 0.63 6.7 37 4.02 6.38 n.a. Amado et al. (2016) Xanthomonas campestris Batch bioreactor Sucrose YE + Pep from fish 2.50 7.0 30 23.20 9.28 0.58 Wang et al. (2016b) Glycerol by-product 4.00 11.00 2.75 0.28 Batch bioreactor Hydrolyzed kitchen Waste Pep 3.40 7.0 30 11.73 3.45 n.a. Li et al. (2016b) Shake flask Cheese whey YE + Pep 3.00 7.20 2825 25.00 8.33 n.a. Silva et al. (2009) Zymomonas mobilis Batch bioreactor Sucrose YE 2.0 5.0 28 15.52 7.72 n.a Silbir et al. (2014) Corn steep liquor 13.91 6.96 Malt 10.30 5.15 ) SO 1.00 n.a. 25 15.16 15.46 0.66 Oliveira et al. (2007) Shake flask Sugar cane syrup YE + (NH4 2 4 F. Freitas et al. / Bioresource Technology 245 (2017) 1674–1683 1679

productivity, as well as on the composition of the synthesized polysaccharides (Freitas et al., 2011a). Moreover, the cost benefit achieved by the use of low-value substrates might be lost if the downstream processing becomes more costly to guaranty the pur- ity of the final product (Seviour et al., 2011). Other elements, such as phosphorus, potassium and metal cations, are also required for microbial growth and EPS synthesis. Phosphorus is an important element for secondary metabolism, Survase et al. (2007b) and it also regulates lipid and uptake by the cells. and Moreover, those salts also serve as sources of potassium or magne- sium, which is a cofactor for some enzymes, required in the carbo- hydrate metabolism and in many transport processes (Survase et al., 2007a,b). The addition of precursors, such as nucleotide phosphate sugars

Wu et al. (2016) Cheng et al. (2011) Özcan et al. (2014) Wang et al. (2015) Sharma et al. (2013) Göksungur et al. (2011) andWang Wu et et al. al. (2014) (2016) Choudhury et al. (2012) Papinutti (2010) Mohtar et al. (2016) Fazenda et al. (2010) Mohtar et al. (2016) Tang et al. (2009) Desai et al. (2008) Farina et al. (2009) Taskin et al. (2010) Survase et al. (2007a) Survase et al. (2007a) or amino acids, were reported to enhance EPS synthesis. For exam- ple, Survase et al. (2007b) reported that the use of the sugar nucleotides UMP and UDPG, as well as the amino acid L-lysine, (g/g) References were effective in improving scleroglucan production, though hav- p/s Y ing a negative impact on the polymer’s Mw. Lysine and arginine are essential for Streptococcus cell growth and HA production ) (YE, yeast extract; ME, malt extract; Pep, peptone).

p/s because the bacterium is unable to synthetize them (Liu et al., Y

(g/L.day) 2011). UDPGlc and aminoacids like methionine have a stimulating P r effect on cell growth and BC production by A. xylinum. The addition of water soluble polysaccharides, namely, agar, acetan or alginate, was reported to improve BC production (Chawla et al., 2009).

3.1. Process operation conditions ) and yield on substrate ( C) EPS (g/L) P r ° Microorganisms are influenced by environmental changes that affect enzyme activity (inhibition or stimulation), protein synthesis (induction and repression), cell morphology, etc. (Survase et al., 2007a,b). Hence, to assure a stable and reproducible bioprocess performance, cultivation parameters, such as pH, temperature, DO concentration and stirrer speed, are often monitored and/or controlled within defined ranges. Mixing and aeration are also relevant parameters as they deter- 4–775.4 5.5–6.53 28–30 2.0–5.0 7.54–4.7 23.1–31.422 30 6.8 3.30–7.86 5.5–7.26 28 28 0.52 23.1 30 3.8 39.2 19.2–36.17 4.09–9.04 39.8 3.30 28 n.a. 3 7.312 22.2 n.a. 13.27 4.5 0.40 4.5 0.80 11.10 28 30 0.49 16.22–21.8 26.0 5.41–7.27 0.33–0.37 mine 13.0 the 0.74 availability of nutrients and oxygen. Vigorous agitation and/or aeration have been reported to lead to high EPS production 4 4 4 4 4 4 by some microorganisms. Examples include HA (Liu et al., 2008a) SO SO SO SO SO SO 3 3 2 2 2 2 2 2 ) ) ) ) ) )

4 4 4 4 4 4 and gellan (Prajapati et al., 2013a). Nevertheless, for alginate (Cimini et al., 2012) and FucoPol (Freitas et al., 2011b), production is favored by low DO levels. High agitation rates may cause damage to the cells or alter the physical-chemical properties of the synthe- sized polymers. For instance, high stirring speed caused a reduc- tion of the Mw of alginate produced by A. vinelandii (Galindo et al., 2007), while xanthan production was impaired due to cell damage caused by high agitation speed (Palaniraj and Jayaraman, 2011). In contrast, low agitation may not provide adequate mixing and, consequently, compromise the mass transfer of nutrients and oxygen through the culture. For example, at lower stirring rates, xanthan production was reduced, causing viscosity increase and then lower oxygen mass transfer.

3.2. Cultivation mode

At lab scale, batch shake flask cultures are usually used initially to define nutritional requirements and culture conditions. In shake Shake flask Sucrose YE; (NH Fed-batch bioreactorAir-lift bioreactor SucroseFed-batch bioreactorBatch bioreactor Glucose SucroseShake flaskShake flask GlucoseShake flaskShake flaskBatch YE; Potato bioreactor (NH hydrolysateFed-batch Rice bioreactor hull YE; hydrolysate (NH Repeated YE; Jatropha fed-batch Glucose YE; (NH seed (NH cake GlucoseFed-batch bioreactor Glucose Glucose YE;Shake (NH Corn flask steep liquor YE; 5 PepShake flaskShake flaskShake flask Sucrose YE; 4.5 Pep YE 5 ME YE Waste loquat kernels YE; Pep Sugarcane juice/molasses 20 Coconut water n.a. n.a. 5 6.0 88.59 10 18 YE; NaNO 20 5 28 4.0 n.a. 17.72 3 4.0 3.0–5.5 83.9 3 3.5 30 4.0 30 0.60 30 4.5 n.a. 4.55 2.59–3.54 16.78 5 30 3 8.1 10.5 0.14–0.20 28 6.61 0.7 n.a. 0.91 28 4.5 19.21–23.87 0.52 0.81 6.40–7.96 12.08 1.32 n.a. 28 n.a. n.a. 0.89 4.03 12.58 1.03 n.a. 4.19 n.a Batch bioreactor Sucrose YE; NaNO flasks, however, it is not possible to monitor or control parameters, which greatly affect EPS synthesis in many microorganisms. In view of this, the screening process should ideally be performed in small bioreactors with similar design as those of the large scale production fermenters. The selection of the most adequate cultiva- tion mode will depend on whether EPS production is growth asso- ciated (e.g. gellan) or non-growth associated (e.g. curdlan). Most Aureobasidium pullulans Ganoderma lucidum Sclerotium rolfsii Microorganism Process Carbon source Nitrogen source Time (d) pH T ( microbial EPS production processes are simple batch cultures or Table 3 EPS production by some fungi: carbon and nitrogen sources, cultivation conditions and EPS production, volumetric productivity ( single pulse fed-batch cultures, following exhaustion of nitrogen 1680 F. Freitas et al. / Bioresource Technology 245 (2017) 1674–1683 source in the medium (Seviour et al., 2011). These processes are the type of polysaccharide and the desired degree of purity. The simple and usually result in high substrate to product yields. cultivation broth can simply be dried, thus yielding a crude pro- Nevertheless, other cultivation modes were proposed, including duct that may find use in some applications but is not adequate fed-batch and continuous culture. Although continuous culture to high-value areas. Therefore, most commonly, the downstream systems usually achieve higher productivities, there is an increased processing involves several steps, starting with cell removal by risk of contamination and the possibility of the development of centrifugation or filtration, followed by recovery of the polymer lower yielding genetic variants (Seviour et al., 2011). The repeated from the cell-free supernatant. A frequently used procedure for fed batch fermentation strategy, in which a portion of the culture is the later step is the precipitation of the polymer by addition of a periodically replaced by fresh medium, can reduce the overall cul- water-miscible non-polar solvent, such as acetone, ethanol or iso- tivation time and avoids catabolite repression. This strategy has propanol. The precipitate can then easily be separated from the been implemented, for example, for the production of EPS by the solvent-water mixture and dried. Several additional procedures fungus G. lucidum (Mohtar et al., 2016) and for levan production can be used to remove contaminants, namely re-precipitation with by B. subtilis (Shih et al., 2010). diluted aqueous solutions, deproteinization by chemical or enzy- matic methods and membrane processes. The downstream process 3.3. Bioreactor design should be chosen based on the purity demand for the desired application. For instance, in the case of FucoPol using acetone pre- Stirred tank reactors (STRs) are the most utilized fermenters at cipitation the inorganic content was 32.5%, which was reduced to both lab and industrial scale. The two most commonly used fer- zero by using a dialysis membrane of 10,000 MWCO membrane menter configurations for microbial cultivation are the continuous (Freitas et al., 2011b). STR (CSTR) and the air-lift reactor (ALR). In CSTR, radial flow impel- lers (e.g. Rushton turbine impellers) create turbulence that assure homogeneous mixing of the broth, promoting heat, oxygen and 4. Bioprocess development and scale-up nutrient mass transfer to the cells. However, as the agitation increases, so does the shear stress that can impact on the product’s For bioprocess development, each producer must be subjected quality. To work under low-shear conditions, ALR are used in to studies for optimization of culture parameters. Focus must be which air is injected through a sparger placed at the bottom of given to the reduction of the synthesis of unwanted by-products the reactor. The broth has lower density at the bottom and it that would decrease EPS yield and productivity. To reach high becomes denser as it raises to the top of the vessel; then, it des- yields of EPS with the required purity and properties, medium cends through a downcomer or external loop (Castillo et al., 2015). composition, cultivation conditions, cultivation mode and bioreac- Other fermenter configurations have been used for EPS produc- tor design must be optimized. The choice of the parameters to be tion by different microorganisms. For example, continuous produc- optimized, as well as the range over which they will be evaluated, tion of levan in a packed-bed bioreactor containing Ca-alginate are of the outmost relevance. immobilized Z. mobilis B-14023 showed that the system was stable Statistically based experimental design methods are often used and efficient with low input and production costs. However, in to evaluate possible interactions between each operating condi- long term fermentations occurs a pressure drop and rupture of tion, instead of the conventional one variable at a time method, Ca-alginate gel beads (Silbir et al., 2014). which is laborious and time consuming. One of the most frequently For BC production, other fermenter configurations have been used statistical-based approach for optimization of bioprocesses is developed because under stirred conditions the produced polymer the response surface methodology (RSM), in which several factors gets stuck to the reactors shaft. Therefore, more effective agitated are simultaneously tested in a reduced number of experiments. cultures reactor have been designed and evaluated, namely spher- The effect of the different tested factors are evaluated and models ical type bubble column and modified air lift reactor. Besides are derived that allow predicting the optimal conditions in a given achieving higher productivities, BC recovery and reactor cleaning process (Desai et al., 2008). This approach has been used for opti- was facilitated. Other configurations were also tested for BC pro- mization of EPS production by many microorganisms, including duction, such as the rotary disks reactor, rotary biofilm contactor, the production of pullulan (Göksungur et al., 2011; Mehta et al., aerosol bioreactor and horizontal lift bioreactor. 2014), xanthan (Psomas et al., 2007), levan (Srikanth et al., The bioreactor cultivation of thermophiles poses some specific 2015b) and FucoPol (Torres et al., 2012). Another empirical method difficulties. The lower solubility of oxygen at higher temperature for bioprocesses optimization is the use of artificial intelligence- determines that good aeration and agitation must be provided. based black box approach, namely, artificial neural networks Moreover, high investments are required for air compressors and (ANN). Desai et al. (2008) have compared both strategies as tools heat exchangers, and for the fermenters that must be especially for the optimization of scleroglucan production by S. rolfsii and built to withstand the elevated temperatures they will be exposed concluded that, while RSM was more useful to get information to. The advantages of using thermophilic microorganisms include on the interactions between the different factors, ANN was more their high cell growth rates at elevated temperatures that trans- accurate in finding optimal conditions for maximum yield. lates into short cultivation times, lower risk of contamination, good As a consequence of increased cell growth, changes in cell mor- mass transfer and lower viscosity of the broth. Considering the low phology and/or accumulation of EPS in the extracellular environ- levels of EPS synthesis and their higher production costs, the inter- ment, in most microbial EPS production processes there is est in developing bioprocesses based on thermophilic microorgan- usually an increase of the broth’s apparent viscosity that may isms would be driven by their unique physical-chemical and increase several orders of magnitude, and, often, a change to shear biological properties, for use in high-value market niches where thinning fluid behavior. Such changes in broth rheology affect the the desired properties or the degree of purity are not fulfilled by mass and heat transfer in the bioreactor, as well as shear stress and the traditional polymers (Kambourova et al., 2016). mixing efficiency (Fazenda et al., 2010; Seviour et al., 2011). The scale-up of processes for the production of microbial EPS is 3.4. Downstream processing challenging because many factors must be taken into considera- tion, especially the fermenter’s hydrodynamics due to the changing The specific method used for recovery of EPS from the cultiva- rheological properties of the broth during cultivation. As the pro- tion broth depends on characteristics of the producing organisms, cess scale moves from lab to production level, the heterogeneities F. Freitas et al. / Bioresource Technology 245 (2017) 1674–1683 1681 increase with reactor volume as a result of the increased physical the productivity and lower production costs, but mainly on poly- distance between the impellers and vessel walls, and the mer characterization and the proof-of-concept of their application corresponding decreased specific power inputs. To obtain high in high-value pharmaceutical and cosmetic areas, where product and consistent polymer yields, it is imperative to standardize the quality and functional properties are far more relevant than pro- large-scale production processes, under controlled conditions, con- duction cost. Hence, the greatest potential for development of sidering the producing strain. Monitoring and control of the EPS novel microbial EPS is on those high-value market niches. production processes are essential to guaranty consistency of pro- duct yield and quality, as well as to comply with regulatory requirements. The methods developed and used by the industry Acknowledgements to monitor and control fermentation processes cannot easily be implemented in processes for the production of microbial EPS This work was supported by the Unidade de Ciências Biomolec- because it is difficult to link key process factors. Hence, although ulares Aplicadas (UCIBIO), which is financed by national funds some real-time monitoring techniques have been attempted, most from FCT/MEC (UID/Multi/04378/2013) and co-financed by the often EPS production monitoring relays on off-line analytical meth- ERDF, under the PT2020 Partnership Agreement (POCI-01-0145- ods that required removing samples from the bioreactor (Seviour FEDER-007728). Cristiana A. V. Torres acknowledges FCT/MEC for et al., 2011). Post-Doctoral fellowship SFRH/BPD/87774/2012.

5. Bottlenecks and future prospects References

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